Crotalase, a Fibrinogen-Clotting Snake Venom Enzyme: Primary Structure and Evidence for a Fibrinogen Recognition Exosite Different from Thrombin

Summary

Crotalase, a fibrinogen-clotting enzyme isolated from the venom of Crotalus adamanteus, and its overlapping fragments were subjected to Edman degradation. The resulting amino acid sequence, VIGGDEC NINEHRFLVALYDYWSQLFLCGGTLINNEWVLTAAHCDRTHI LIYVGVHDRSVQFDKEQRRFPKEKYFFDCSNNFTKWDKDIM LIRLNKPVSYSEHIAPLSLPSSPPIVGSVCRAMGWGQTTSPQET LPDVPHCANINLLDYEVCRTAHPQFRLPATSRTLCAGVLEG GIDTCNRDSGGPLICNGQFQGIVFWGPDPCAQPDKPGLYTK VFDHLDWIQSIIAGEKTVNCP, is characteristic of a serine protein-ase. Comparison with thrombin, the physiological fibrinogen-clotting enzyme, showed that thrombin’s fibrinogen-recognition exosite (FRE) is poorly represented in crotalase. Hirudin, a FRE-dependent inhibitor, had no effect on crotalase. Spatial modeling of crotalase yielded a possible alternative fibrinogen-recognition site comprised of Arg 60F, Lys 85, Lys 87, and Arg 107 (underlined in the sequence above). Crotalase also lacks thrombin’s YPPW loop, as well as its functionally important ETW 146-148, and its heparin-binding site. The enzyme contains a single asparagine-linked glycosylation site, NFT, bearing neutral and amino sugars that account for 8.3% of the enzyme’s total molecular weight of 29,027. The calculated absorbance of crotalase at 280 nm, 1%, cm^-1is 15.2.

 

• References

• 1 Markland FS, Damus PS. Purification and properties of a thrombin-like enzyme from the venom of Crotalus adamanteus (eastern diamondback rattlesnake). J Biol Chem 1971; 246: 6460-73.
• 2 Markland FS, Pirkle H. Thrombin-like enzyme from the venom of Crotalus adamanteus (eastern diamondback rattlesnake). Thromb Res 1977; 10: 487-94.
• 3 Pirkle H. Thrombin-like enzymes from snake venoms: An updated inventory. Thromb Haemost 1998; 79: 675-83.
• 4 Massova I, Pirkle H, Edwards BFP, Mobashery S. Insights into the three-dimensional structure of crotalase: Implications for biological activity and substrate specificity. Bioorg Med Chem Lett 1997; 7: 3139-44.
• 5 Bajwa WW, Markland Jr. FS. A new method for purification of the thrombin-like enzyme from the venom of the eastern diamondback rattlesnake. Thromb Res 1979; 16: 11-23.
• 6 Pirkle H, Theodor I, Miyada D, Simmons G. Thrombin-like enzyme from the venom of Bitis gabonica. Purification, properties, and coagulant actions. J Biol Chem 1986; 261: 8830-5.
• 7 Hirs CHW. Reduction and S-carboxymethylation of proteins. Methods Enzymol 1967; 11: 199-203.
• 8 Habeeb AFSA. Reaction of protein sulfhydryl groups with Ellman’s reagent. Methods Enzymol 1972; 25: 457-64.
• 9 Habeeb AFSA, Cassidy HG, Singer SJ. Molecular structural effects produced in proteins by reaction with succinic anhydride. Biochim Biophys Acta 1958; 29: 587-93.
• 10 Fontana A, Dalzoppo D, Grandi C, Zambonin M. Cleavage at tryptophan with o-iodosobenzoic acid. Methods Enzymol 1983; 91: 311-7.
• 11 Huber R, Bode W. Structural basis of the activation and action of trypsin. Acc Chem Res 1978; 11: 114-22.
• 12 Itoh N, Tanaka N, Mihashi S, Yamashina I. Molecular cloning and sequence analysis of cDNA for batroxobin, a thrombin-like snake venom enzyme. J Biol Chem 1978; 262: 3132-5.
• 13 Nikai T, Ohara A, Komori Y, Fox JW, Sugihara H. Primary structure of a coagulant enzyme, bilineobin, from Agkistrodon bilineatus venom. Arch Biochem Biophys 1995; 318: 89-96.
• 14 Pirkle H, Theodor I. Thrombin-like enzymes. In: Snake Venom Enzymes.
• 15 Bailey GS. (ed) Alaken, Inc.; Fort Collins, Colorado: 1998: 39-69.
• 16 Bode W, Turk D, Karshikov AJ. The refined 1.9-Å X-ray structure of D-Phe-Pro-Arg chloromethylketone-inhibited α-thrombin: Structure analysis, overall structure, electrostatic properties, detailed active-site geometry, and structure-function relationships. Protein Sci 1992; 1: 426-71.
• 17 Kornfeld R, Kornfeld S. Assembly of asparagine-linked oligosaccharides. Ann Rev Biochem 1985; 54: 631-64.
• 18 Tanaka N, Nakada H, Itoh N, Mizuno Y, Takanishi M, Kawasaki T, Tate S-I, Inagaki F, Yamashina I. Novel structure of the N-acetylgalactosamine containing N-glycosidic carbohydrate chain of batroxobin, a thrombin-like snake venom enzyme. J Biochem 1992; 112: 68-74.
• 19 Lochnit G, Geyer R. Carbohydrate structure analysis of batroxobin, a thrombin-like serine protease from Bothrops moojeni venom. Eur J Biochem 1995; 228: 805-16.
• 20 Pfeiffer G, Dabrowski U, Dabrowski J, Stirm S, Strube KH, Geyer R. Carbohydrate structure of a thrombin-like serine protease from Agkistrodon rhodostoma. Structure elucidation of oligosaccharides by methylation analysis, liquid secondary-ion mass spectrometry and proton magnetic resonance. Eur J Biochem 1992; 205: 961-78.
• 21 Pace CN, Vajdos F, Fee L, Grimsley G, Gray T. How to measure and predict the molar absorption coefficient of a protein. Protein Sci 1995; 4: 2411-23.
• 22 Gill SC, von Hippel PH. Calculation of protein extinction coefficients from amino acid sequence data. Anal Biochem 1989; 182: 319-26.
• 23 Bode W, Mayr I, Baumann U, Huber R, Stone SR, Hofsteenge J. The refined 1,9-Å crystal structure of human α-thrombin: interaction with D-Phe-Pro-Arg chloromethylketone and significance of the Tyr-Pro-Pro-Trp insertion segment. EMBO J 1989; 8: 3467-73.
• 24 Le Bonniec BF, Guinto ER, MacGillivray RTA, Stone SR, Esmon CT. The role of thrombin’s Tyr-Pro-Pro-Trp motif in the interaction with fibrinogen, thrombomodulin, protein C, antithrombin III, and the Kunitz Inhibitors. J Biol Chem 1993; 268: 19055-61.
• 25 Le Bonniec BF, Guinto ER, Esmon CT. Interaction of thrombin deS-ETW with antithrombin III, the Kunitz inhibitors, thrombomodulin and protein C. Structural link between the autolysis loop and the Tyr-Pro-Pro-Trp insertion of thrombin. J Biol Chem 1992; 267: 19341-8.
• 26 Dang QD, Sabetta M, Di Cera L. Selective loss of fibrinogen clotting in a loop-less thrombin. J Biol Chem 1997; 272: 19649-51.
• 27 Martin PD, Robertson W, Turk D, Huber R, Bode W, Edwards BFP. The structure of residues 716 of the Aα-chain of human fibrinogen bound to bovine thrombin at 2.3-Å resolution. J Biol Chem 1992; 267: 7911-20.
• 28 Damus PS, Markland Jr FS, Davidson TM, Shanley JD. A purified procoagulant enzyme from the venom of the eastern diamondback rattlesnake (Crotalus adamanteus): In vivo and in vitro studies. J Lab Clin Med 1972; 79: 906-23.
• 29 Fenton JW I I, Bing DH. Thrombin active-site regions. Sem Thromb Hemost 1986; 12: 200-8.
• 30 Stubbs MT, Bode W. A player of many parts: The spotlight falls on thrombin’s structure. Thromb Res 1993; 69: 1-58.
• 31 Chang JY. Deciphering the structural elements of hirudin C-terminal peptide that bind to the fibrinogen recognition site of α-thrombin. Biochemistry 1991; 30: 6656-61.
• 32 Rydel TJ, Tulinsky A., Bode W, Huber R. The structure of a complex of recombinant hirudin and human α-thrombin. J Mol Biol 1991; 221: 583-601.
• 33 Vitali J, Martin PD, Malkowski MG, Robertson WD, Lazar JB, Winant RC, Johnson PH, Edwards BFP. The structure of a complex of bovine α-thrombin and recombinant hirudin at 2.8-Å resulution. J Biol Chem 1992; 267: 17670-8.
• 34 Coughlin SR, Vu T-KH, Hung DT, Wheaton I. V. Characterization of a functional thrombin receptor. Issues and opportunities. J Clin Invest 1992; 89: 351-5.
• 35 Binnie CG, Lord ST. A synthetic analog of fibrinogen a 27-50 is an inhibitor of thrombin. Thromb Haemost 1991; 65: 165-8.
• 36 Hartley BS, Kauffman D. Corrections to the amino acid sequence of bovine chymotrypsinogen A. Biochem J 1966; 101: 229-31.
• 37 Swift GH, Dagorn J-C, Ashley PL, Cummings SW, MacDonald RJ. Rat pancreatic kallikrein mRNA: Nucleotide sequence and amino acid sequence of the encoded preproenzyme. Proc Natl Acad Sci USA 1982; 79: 7263-7.
• 38 Walsh KA, Neurath H. Trypsinogen and chymotrypsinogen as homologous proteins. Proc Natl Acad Sci USA 1964; 52: 884-9.
• 39 Magnusson S, Peterson TE, Sottrup-Jensen L, Claeys H. Complete primary structure of prothrombin: Isolation, structure and reactivity of ten carboxylated glutamic acid residues and regulation of prothrombin activation by thrombin. In: Proteases and Biological Control. Reich E, Rifkin DB, Shaw E. (eds) Cold Spring Harbor Laboratory, Cold Spring Harbor; New York; 1975: pp 123-49.
• 40 Pirkle H, Theodor I, Henschen A. Crotalase, a fibrinogen-clotting venom enzyme: Primary structure and evidence for lack of a fibrinogen recognition exosite homologous to that of thrombin. Haemostas 1996; 26 (Suppl. 03) Suppl 452 (Abstract)
• 41 Burkhart W, Smith GFH, Su J-L, Parikh I, LeVine H I. II. Amino acid sequence determination of Ancrod, the thrombin-like α-fibrinogenase from the venom of Akistrodon rhodostoma . FEBS Lett 1992; 297: 297-301.
• 42 Magalhães A, Da Fonseca BCB, Diniz DR, Gilroy J, Richardson M. The complete amino acid sequence of a thrombin-like enzyme/gyroxin analogue from venom of the bushmaster snake (Lachesis muta muta). FEBS Lett 1993; 329: 116-20.
• 43 Nishida S, Fujimura Y, Miura S, Ozaki Y, Usami Y, Suzuki M, Titani K, Yoshida E, Sugimoto M, Yoshioka A, Fukui H. Purification and characterization of bothrombin, a fibrinogen-clotting serine protease from the venom of Bothrops jararaca . Biochemistry 1994; 33: 1843-9.
• 44 Shieh T-C, Kawabata S-I, Kihara H, Ohno M, Iwanaga S. Amino acid sequence of a coagulant enzyme, flavoxobin, from Trimeresurus flavoviridis venom. J Biochem 1988; 103: 596-605.
• 45 Hahn B-S, Yang K-Y, Park E-M, Chang I-M, Kim Y-S. Purification and molecular cloning of calobin, a thrombin-like enzyme from Agkistrodon ca-liginosus (Korean viper). J Biochem 1996; 119: 835-43.